Jared Head February 12, 2007 Biology 107 Properties of Enzymes and the Factors that Affect Them Introduction Within our bodies, thousands of complex biological processes are constantly taking place. We require material transport, energy synthesis, and the manufacturing of various proteins, hormones, and other molecules. Almost all of these everyday processes rely on the function of enzymes to take place. Enzymes are catalysts; they are chemical agents that “speed up a reaction without being consumed by the reaction” (Campbell and Reece 150). Enzymes are specifically grouped according to their function, and this information can often provide us with clues regarding what type of reaction that enzyme will catalyze. An enzyme can only react with a very specific type of substrate, which binds to the area called the “active site”. After the substrate binds to the active site, an enzyme-substrate complex is created, and then the products are released. The enzyme is not consumed in the reaction and is thus able to be reused. Both active sites and substrates have very specific shapes, which is why enzymes will only catalyze reactions when they interact with a specific substrate. The function of enzymes can be affected by a variety of factors, including temperature, pH, and enzyme inhibitors, which work by binding to the active site before the substrate can. Because the activity and functionality of enzymes can be measured by the coloration of the end product, the results of these experiments can be quantified by measuring absorbance with a spectrophotometer. In
this lab, we will investigate how the aforementioned factors can affect the ability of enzymes to catalyze various reactions. Materials and Methods In part one of the experiment, five test tubes were prepared according to Table 1 (see appendix) with varying concentrations of stock 0.25 mM p-nitrophenol solution. A spectrophotometer was used to measure the absorbance of these solutions, allowing us to quantify the data and generate a standard curve for the amount of end product, p- nitrophenol. Using these values, the amount of p-nitrophenol (in micromoles) was calculated and used to create a standard curve. In the next part of the experiment, three test tubes were prepared according to Table 2 (see appendix) with varying amounts of the enzyme, acid phosphatase and different amounts of substrate in each tube. Two ml of 0.2M sodium hydroxide (NaOH) were then added to each tube to stop the reaction and visually enhance the color of p-nitrophenol, the end product. To test the effect on an inhibitor on the activity of acid phosphatase , two tubes were prepared in accordance with Table 3 (see appendix). After pipetting the correct amount of pH buffer and the inhibitor Na 2 PO 4 into each tube, 0.25 ml of the substrate, p-nitrophenyl phosphate was added to both tubes. Again, 2 ml of 0.2M NaOH were added and the absorbance was determined using the spectrophotometer.